Antimicrobial susceptibility testing

AST was performed in accordance with CLSI M07-A10 guidelines for BMD¹⁴. Briefly, bacterial cell suspensions equivalent to a 0.5 McFarland standard were prepared by emulsifying colonies from fresh sub-cultures in 0.9% (w/v) NaCl and verified using an Oxoid Turbidimeter (Oxoid Canada, ON, CA). The same 0.5 McFarland suspension was used to create the respective starting inoculum for the D300e or rBMD methods on the same day and was diluted in CAMHB to an appropriate intermediate concentration in order to achieve a final inoculum density of 5 × 10⁵ CFU/mL per well (100 µL), as confirmed by colony counts. The diluted inoculum was added to each plate with a multichannel pipette and test panels were incubated statically at 35ºC ± 2 °C for 20 to 24 h. The MIC for each antimicrobial-organism combination was assessed visually against a black background by a single reader and reported as the lowest concentration of the agent that completely inhibited isolate growth. Isolates were numerically coded to blind the reader to the identity of the test isolates. The corresponding MICs for each antimicrobial/organism combination were determined using CLSI 2020 breakpoints³.